Gene expression of bacteria regulated by numerous mechanisms which leads to the production of specific or group of genes. During stress condition genes shows response to environmental stimuli. E.coli cells defence itself in oxidative condition by DNA repair mechanisms and also several regulators including OxyR, SoxRS and RpoS which plays an important role. In oxidative stress OxyR undergo conformational changes to avoid oxidative lesions and also control the expression of sufA as well as ahpC genes.
In our experiment, rapidly growing E.coli was treated with oxidative stress (H2O2). Transcription is the first step leading to gene expression that is the reason we isolate RNA at different time intervals. Isolated RNA concentration from stressed cells were measured using nanodrop and RNA concentration was gradually increased with time (t0 t20) (table 2). It revealed more expression of genes. RNA can be degrade or contaminate very easily by mishandling, prolong storage or more on. Nanodrop result showed the RNA samples was in the range of purity. RT-PCR result showed the expression level of sufA and ahpC gene (rpoA as refernce) under stressed condition. H2O2 causes several injuries to E.coli, so to protect itself, it activate OxyR regulon which induce sufA operon and ahpC. Expression of sufA as well as ahpC degrade the H2O2 activity and give a resistance mechanism to E.coli. Melting curve plot shows expected behaviour (peak) of target genes. No other peak (may be for primer dimer) was shown which proved that the RT PCR was successfully done. Upregulation of both genes were observed. In sufA gene upregulation was observed around 361.90 fold at t5 and 131.74 fold at t20 whereas upregulation of ahpC was observed 9.91 and 41.54 fold at t5 and t20 respectively. It also noted that fold change of untreated (t0) samples just 1, which means no change in expression level.
In second part of in vivo experiment, amplification of sufA and ahpC gene promoter was successful and we observed the band at expected position (PsufA-395bp and PahpC-344bp).Later the ligation reaction shows some unexpected results which revealed that our plasmid and gene of interest was not ligated. And might be in some cases gene of interest binds itself and shows bands. Follow through our transformation experiment also failed. We observed few colonies on T1, T2, T3 plates it might be due to the escapes of plasmids. In transformation LB media with AmpR was used. AmpR kills bacteria by preventing the cell wall synthesis. So it was used as selectable marker. Only E.coli with transformed Because of transformation failure in fluorescence microscopy experiment we used only T4 (Undigested plasmid) construct instead of T1 and T2. T4 plasmids carried AraC gene which degrade arabinose and shows brighter GFP signal in arabinose containing LB media and control (no arabinose) didn’t shows any GFP signal (araC gene was not active).
Transformation was not successful it depends on few assumptions, first, it might be just one restriction enzyme worked and plasmid self-ligated. That reason in ligation experiment we observed the band of our gene of interest at same position of their original size. Ligation experiment of pBADsfGFPx1+sufA showed star activity (may be restriction enzyme was not deactivate) of restriction enzymes might be thatswhy we observed more band on gel electrophoresis. Second, the size of our empty plasmid vector and our construct vectors we can say almost the same. That means there was no gene of interest in the plasmids construct. Third, might be the ligation solution was not good enough to ligate our product properly. So those might be leads the failure of ligation reaction as well as our in vivo experiment